Antibiotic hyper-resistance in a class I aminoacyl-tRNA synthetase with altered active site signature motif

Antibiotics target key biological processes that include protein synthesis. Bacteria respond by developing resistance, which increases rapidly due to antibiotics overuse. Mupirocin, a clinically used natural antibiotic, inhibits isoleucyl-tRNA synthetase (IleRS), an enzyme that links isoleucine to its tRNAIle for protein synthesis. Two IleRSs, mupirocin-sensitive IleRS1 and resistant IleRS2, coexist in bacteria. The latter may also be found in resistant Staphylococcus aureus clinical isolates. Here, we describe the structural basis of mupirocin resistance and unravel a mechanism of hyper-resistance evolved by some IleRS2 proteins. We surprisingly find that an up to 103-fold increase in resistance originates from alteration of the HIGH motif, a signature motif of the class I aminoacyl-tRNA synthetases to which IleRSs belong. The structural analysis demonstrates how an altered HIGH motif could be adopted in IleRS2 but not IleRS1, providing insight into an elegant mechanism for coevolution of the key catalytic motif and associated antibiotic resistance.

Supplementary Fig. 4: Generation of an ancestral IleRS phylogenetic tree.a) An ancestral sequence reconstruction was performed by the PAML package 4 using a multiple sequence alignment of the PFAM domains of 379 IleRS sequences (158 HXGH IleRS1, 41 archeal HXG(H/T) IleRS, 77 HXGH IleRS2 and 86 GXHH IleRS2 sequences), and four ValRS sequences, used as an outgroup.The tree is plotted and annotated using the program package ITOL 5 .Naturally occurring reversion of the non-canonical to the canonical motif occurred in the Deinococcus-Thermus clade (red triangle).b) Alignment of ancestral sequences of the selected nodes.Source data (the tree and the alignment) are provided as a Source data file.Supplementary Fig. 5: Electron density and OMIT maps of Ile-AMS bound to PmIleRS1 and PmIleRS2.a) 2Fo-Fc Fourier difference map (blue) of the wt-HVGH-PmIleRS2:Ile-AMS complex.The map is resolved at 2.2 Å and contoured at a 2σ level.The unbiased Fo-Fc Fourier difference OMIT map, contoured at 4σ (green) and -4σ (red) levels, shows the presence and conformation of the isoleucyl-adenylate analogue (Ile-AMS).b) The 2Fo-Fc and corresponding Fo-Fc OMIT Fourier difference density maps of the mut-GVHH-PmIleRS2:Ile-AMS unambiguously reveal the mutated signature motif residues and the bound Ile-AMS analogue.The map is resolved at 2.3 Å, with colors and contour levels as above.c) 2.9 Å 2Fo-Fc Fourier difference density and corresponding Fo-Fc OMIT maps of the wt-HMGH-PmIleRS1:Ile-AMS complex, contoured at 2σ and +/-2σ, respectively, and colored as above.d) The 2Fo-Fc and corresponding Fo-Fc OMIT Fourier difference density maps of the mut-GMHH-PmIleRS1:Ile-AMS unambiguously reveal the mutated signature motif residues and the bound Ile-AMS analogue.The map is resolved at 3.2 Å, with contour levels of 2σ for the 2Fo-Fc map and +/-2σ levels for the respective Fo-Fc OMIT maps.For generating the unbiased Fo-Fc Fourier difference OMIT maps, the cofactors were removed from the models, followed by refinement until convergence (5 cycles) in Phenix after setting the B values to 100 Å 3 (a, c) in an overall similar mode as to the corresponding mutants (b, d).However, in the active site of the catalytically inactive mut-GMHH-PmIleRS1 (b), the adenine base is mispositioned due to a steric constraint.See Fig. 4 and main text for further details.Supplementary Fig. 8: The HXGH motif displacement is not dictated by a steric clash with a nearby α2 helix residue.a, c, d, e) Structural analysis identifies the α2 helix position (W130) that is in close proximity to the 4 th His of the HXGH motif.A sequence alignment highlights this position as bulky in IRS2 (W130) but not in IRS1 (Q136), suggesting a crucial role of W130 in the displacement of the HXGH motif in IleRS2.However, in the structure of the W130Q PmIleRS2 mutant bound to Ile-AMS, the HXGH motif is at the same position of as in the WT enzyme (i.e. it remained replaced relative to IRS1:Ile-AMS).Kinetic analysis reveals that W130Q PmIleRS2 mutant displays an 18-fold lower kcat relative to the wild-type with a minor effect on the KM for Ile or ATP.The Ki for mupirocin remained the same as for the wild-type enzyme.Source data are provided as a Source Data file.Data are presented as the average value ± SD of three independent experiments.b) A 2Fo-Fc Fourier difference map of the W130Q-HVGH-PmIleRS2:Ile-AMS complex, resolved at 2.3 Å and contoured at a 2σ level, is shown in blue.The unbiased Fo-Fc Fourier difference OMIT map is contoured at +/-4σ (green/red), revealing the presence and conformation of the Ile-AMS analogue.For generating the unbiased Fo-Fc Fourier difference OMIT map, the cofactor was removed from the model, followed by refinement until convergence (5 cycles) in Phenix after setting the B values to 100 Å 3  The map is resolved at 1.9 Å and contoured at a 2σ level.The unbiased Fo-Fc Fourier difference OMIT map, contoured at 5σ (green) and -5σ (red) levels, shows the presence and conformation of mupirocin.b) A 2.95 Å 2Fo-Fc Fourier difference density and corresponding Fo-Fc OMIT maps of the wt-HMGH-PmIleRS1:mupirocin complex, contoured at 2σ and +/-2σ, respectively, are colored as above.For generating the unbiased Fo-Fc Fourier difference OMIT maps, the cofactors were removed from the models, followed by refinement until convergence (5 cycles) in Phenix after setting the B values to 100 Å 3 and randomizing the Cartesian coordinates by 0.1 Å. Supplementary Fig. 12: MD simulations reveal the occurrence of a kink in helix α2 in PmIleRS2 independently of the presence of mupirocin or Ile-AMS.a) The comparison between crystal structures of wt-PmIleRS2 bound to mupirocin (left) or Ile-AMS (right) reveals a kink in the α2-helix only in the mupirocin-bound structure.b) Secondary structures of helix α2 during triplicate 360 ns MD simulation time of wt-PmIleRS2 apo structures created by removing mupirocin from the wt-PmIleRS2:mupirocin crystal structure (left) or Ile-AMS from the wt-PmIleRS2:Ile-AMS crystal structure (right).The distortion in helix α2 originally present in the wt-PmIleRS2:mupirocin structure persists during the entire course of the MD simulation.In contrast, the undistorted helix α2 originally found in the wt-PmIleRS2:Ile-AMS crystal structure becomes distorted already after 5-10 ns of the simulation and remains distorted until its end.c) Snapshots along the helix α2 distortion pathway.The distortion observed during the MD simulations might be driven by placement of residue Phe124 into the hydrophobic pocket formed by Ile148, Val 123, Trp 84, Thr 86 and Glu127, and by the loss of the cation-π interaction of Phe124 and Lys128 that is present in the wt-PmIleRS2:Ile-AMS structure.Taken together, the MD simulations suggest that the kink in helix α2 is an inherent feature of PmIleRS2 and is not related to mupirocin binding.It seems plausible to assume that two different conformations of the active site exist in solution and that binding of mupirocin or Ile-AMS pulls the equilibrium towards the conformations found in the crystal structures.Supplementary Fig. 14 In both cases, the catalytic cores align well (right panels), while whole enzymes superimpose poorly due to different orientations of the editing domain and the different topologies of their C-terminal domains (left panels).Helix α2 in the PmIleRS2:mupirocin structure is distorted compared to the one of PmIleRS1:mupirocin.In IleRS2, the closed arrangement of the KMSKS loop forces binding of the nonanoic moiety of mupirocin into a conformation that precludes formation of a hydrogen bond with Lys 597.See Fig. 5 and main text for further details.Total number of atoms 196644 Total number of water molecules 60148 2 was then purified using column chromatography (Rf = 0.51, CHCl3 / MeOH, 3:1).Yield: 318 mg (60%).

NMR and MS characterization of the compounds 1-3
The NMR spectra were recorded at 298 K by means of a Bruker Avance III HD 400 MHz/54 mm ascend spectrometer equipped with a 5 mm PA BBI 1H/D-BB probe head with z-gradient and automated tuning and matching accessory.The 1 H proton spectra were acquired using 64K data points, spectral width of 20 ppm, recycle delay of 1.0 s, and 16 scans (zg30 pulse program).The 13 C spectra were acquired using 64K data points, spectral width of 220 ppm, recycle delay of 2.0 s, and 4096 scans (deptqgpsp pulse program).The data were processed using TopSpin 3.6.2Bruker software package.Sample concentrations were 16.7 mg/ml (10 mg of compound dissolved in 0.6 ml of solvent, DMSO for compounds 1 and 2, D2O for 3).LC-MS/MS analysis was carried out using an Agilent Technologies 1200 series HPLC system equipped with a binary pump, a vacuum membrane degasser, an automated auto-sampler and an injector interfaced with a 6420 triple quadrupole mass spectrometer with an electrospray ionization source (ESI; Agilent Technologies Inc. Palo Alto, CA, USA).The ESI was operated in a positive mode and negative mode, and samples were detected in the total ion current mode.All data acquisition and processing were performed using Agilent MassHunter software.1 mg of sample was dissolved in 1 ml od MeOH/H20 (1:1, v/v) solvent mixture and 20 L was injected into ESI.
and randomizing the Cartesian coordinates by 0.1 Å. Supplementary Fig. 6: Structure of the C-terminal domain of type 2 IleRS.a) In the wt-PmIleRS2:Ile-AMS structure, we were able to build for the first time the full-length C-terminal anticodon-binding domain of type 2 IleRS.b) The domain consists of 3 subdomains.SD1 (helical bundle domain) and SD2 are structurally homologous to the corresponding subdomains in IleRS1.In IleRS2, the SD3 subdomain is homologous to SD2, while in IleRS1, the corresponding domain adopts a topologically unrelated αβ-fold with a zinc-binding motif (not shown).Further, IleRS2 SD2 features an insertion that adopts a βαβ-fold.The gray mesh around the models represents an unbiased composite OMIT map, contoured at a level of 2σ, showing the respective subdomains.c-e) Comparison with the structures of Thermus thermophilus wt-IleRS2:Ile-AMS (1JZQ [https://doi.org/10.2210/pdb1JZQ/pdb]),Candida albicans wt-IleRS2:Ile-AMP (6LDK [https://doi.org/10.2210/pdb6LDK/pdb])and Saccharomyces cerevisiae wt-IleRS2:reveromycin (7D5C [https://doi.org/10.2210/pdb7D5C/pdb])depicts the novel parts of the structure unraveled by this study.Supplementary Fig. 7: Binding of Ile-AMS to wt-PmIleRS1, wt-PmIleRS2 and their signature motif mutants.Ile-AMS binds to the WT enzymes and randomizing the Cartesian coordinates by 0.1 Å. Supplementary Fig. 9: Structural alignment of the wt-PmIleRS1, wt-PmIleRS2 and their signature motif mutants.a-e) Superposition of wt-and mut-PmIleRS1 or wt-and mut-PmIleRS2 full-length structures bound to Ile-AMS or mupirocin.The HXGH/GXHH motifs and bound ligands are highlighted.The cofactor-bound catalytic domains align well, revealing the conformational changes induced by binding of different ligands.A significant rearrangement of both the editing domain and C-terminal subdomains (SD2, SD3) relative to the catalytic domains are observed only for the pairs in b and c. f) The superposition of wt-PmIleRS1 and wt-PmIleRS2 full-length structures shows that their catalytic and editing domains align well, while the C-terminal domains differ in topology and cannot be superimposed.Supplementary Fig. 10: Binding of mupirocin to wt-PmIleRS1 and wt-PmIleRS2.a) 2Fo-Fc Fourier difference map (blue) of the wt-HVGH-PmIleRS2:mupirocin complex.

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Mupirocin binding to IleRS2 repositions the HXGH motif to the conformation observed in IleRS1.The enzymes were superimposed using the backbone coordinates of the full-length structures (left panels) or the HUP catalytic cores (residues 51-174 and 521-635 in IleRS1 and 40-168 and 513-632 in IleRS2, right panels).a) Structural overlay of PmIleRS1:Ile-AMS and PmIleRS2:mupirocin.The HXGH motif of the PmIleRS2:mupirocin complex assumes a similar conformation as the HXGH motif in PmIleRS1:Ile-AMS.Mupirocin binding to PmIleRS2 coincides with a distortion of helix α2, which is not present in PmIleRS1.Compared to PmIleRS1, the KMSKS loop of PmIleRS2 is around 4 Å closer to the active site.b) Structural overlay of PmIleRS1:mupirocin with PmIleRS2:mupirocin.The overall superposition is very similar to the one presented in panel (a).

Table 3 .
Sequences of the primers used in this study.Unhybridized primer regions (overlap extensions) are shown in bold uppercase lettering.Unhybridized mutagenic overhangs are shown as bold lowercase letters.Restriction enzyme recognition sites are shown in underlined uppercase letters.Primer recognition sites are marked in italic uppercase letters.

Table 4 .
Complete description of the MD simulation system setup